The proposed project is a study, with electrophysiological, pharmacological and morphological (EM) methods, of factors that regulate the growth and differentiation of neurons, and especially the formation of appropriate synaptic interaction. Sympathetic neurons are dissociated from the superior cervical ganglia of newborn rats and allowed to develop in cell culture. Grown by themselves almost all the neurons become adrenergic; grown in the presence of certain non-neuronal cells they eventually (3-5 weeks) become predominantly cholinergic. We have devised a method for growing single neurons, with a small number of beating heart myocytes, in microcultures, 300-500 microns in diameter. The neurons establish interactions with the heart cells with high reliability; this provides a sensitive assay for the transmitter function of the neurons. After 2-3 weeks in microculture, some neurons are cholinergic, some are adrenergic, and some are dual-function (i.e., secrete both transmitters). Morphological studies suggest that the neurons start off with an adrenergic bias; we will determine if the young neurons (4-6 days) are functionally adrenergic before they eventually become cholinergic. We will follow the same individual neurons to determine the time of onset, and the duration, of the dual-function state. We will also undertake a study of the distribution of acetylcholine receptors on the surface of the neurons, and of factors that regulate to this distribution. A third part of the project will make use of a new multichamber culture system to study regional effects (i.e., on different parts of the neurons) of factors that influence the neurons' growth and differentiation.